1. Field of the Invention
The invention generally relates to hyaluronidase 2 (HYAL2) inhibition and, more specifically, to molecules that specifically inhibit HYAL2 activity, to methods of identifying HYAL2-specific inhibitors, and to methods for ameliorating a condition associated with the pro-inflammatory activity of HYAL2, including, for example, chronic inflammation or vasculitis, by specifically inhibiting HYAL2 activity and, therefore, HYAL2 mediated generation of pro-inflammatory and pro-angiogenic hyaluronan intermediate fragments.
2. Background Information
Inflammation and necrosis of blood vessels (vasculitis), including arteries, veins, and capillaries, can occur in connection with exposure to infectious agents, mechanical trauma, radiation, or toxins, and in association with immunological responses or disorders. In many cases, however, no etiology can be determined for a vasculitis (Rubin and Farber, xe2x80x9cPathologyxe2x80x9d 3d ed. (Lippincott-Raven 1999); pages 514-520).
Numerous vasculitis disorders have been identified, and have been classified based on the size of the blood vessels that primarily are affected, i.e., small vessel, medium vessel, or large vessel, or based on general similarities of the disorders, e.g hypersensitivity vasculitis, which includes serum sickness and some, but not all, other disorders that involve, at least in part, an undesirable immune response. Despite these attempted classifications of vasculitis disorders, however, there is considerable overlap of signs and symptoms associated with the vasculitis disorders in different classification groups, and, therefore, it has been difficult to prescribe general treatment protocols.
The occurrence of vasculitis has variously been attributed to an involvement of immune mechanisms and to viral infection. A potential involvement of immune mechanisms originally was based on the identification of immune complexes in serum sickness, which was one of the first human immunological disorders associated with vasculitis. However, evidence is lacking for a role of the immune response in most cases of vasculitis. Anti-neutrophil cytoplasmic antibodies (ANCA) have been identified in association with Wegener""s granulomatosis and microscopic polyarteritis, which are small vessel vasculitis disorders. However, it is not clear whether the appearance of ANCA is causal for these disorders or merely an effect that is observable. In fact, the difficulty in classifying and treating vasculitis is due to a lack of understanding of the etiology of the disorder. Thus, a need exists to identify a general etiology of vasculitis such that methods for early diagnosis and prevention or treatment of this disorder can be developed. The present invention satisfies this need and provides additional advantages.
The present invention is based, in part, on the discovery that structurally related hyaluronidase polypeptides have opposing mechanisms of action, including pro-inflammatory or anti-inflammatory activity, such that broad spectrum hyaluronidase inhibitors can cause undesirable effects when administered to an individual. As disclosed herein, the use of agents that specifically inhibit hyaluronidase 2 (HYAL2), which has pro-inflammatory activity, provides a means to ameliorate pathologic conditions associated an undesirable inflammatory response due to generation of an intermediate hyaluronan catabolite by HYAL2.
Accordingly, the present invention relates to a method of identifying an agent that specifically inhibits HYAL2 activity, without substantially affecting the activity of non-inflammatory and anti-inflammatory hyaluronidases. Such a method can be performed, for example, by contacting HYAL2 and hyaluronan (HA) with a test agent, under conditions sufficient for HYAL2 activity, and detecting a decrease in HYAL2 activity. In the same reaction, or in a different reaction, which can be run in parallel, the activity of one or more non-inflammatory hyaluronidases such as HYAL1 or PH20 can be determined to confirm that an agent that inhibits HYAL2 activity has substantially less, if any, effect on the non-inflammatory (or anti-inflammatory) hyaluronidase(s) and, therefore, is a specific inhibitor of HYAL2.
A screening assay of the invention can be performed using cells that express HYAL2, either naturally or due to introduction of a HYAL2 encoding transgene, or using a detergent extract of such cells, including the membrane fraction, which contains the associated HYAL2. Where a cell that is genetically modified to express HYAL2 is used, or a detergent extract of such a cell, the transgene, which encodes HYAL2, can be transiently contained in the cell, or can be stably maintained by integration into the cell genome. Such a stably transfected cell can provide a standardized source of membrane associated HYAL2 useful for performing a screening assay of the invention. A screening assay of the invention also can be practiced using HYAL2 associated with a synthetic membrane, for example, a lipid bilayer or a unilamellar or multilamellar vesicle such as a liposome.
A decrease in HYAL2 activity can be detected directly by measuring a decrease in the amount or rate of generation of the pro-inflammatory 20 kDa intermediate HA catabolite following addition of the test agent, or can be detected indirectly by detecting decreased expression of a reporter gene regulated from a chemokine promoter, which exhibits induced expression in the presence of the 20 kDa intermediate. Such a reporter gene includes the chemokine promoter, for example, a RANTES, promoter, operatively linked to a nucleotide sequence encoding a detectable polypeptide as an enzyme; a fluorescent or luminescent polypeptide; a ligand (or receptor) that specifically binds a particular receptor (or ligand); or the like.
An agent that specifically inhibits HYAL2 activity can be any type of molecule, including, for example, a peptide (or polypeptide), a polynucleotide, a peptidomimetic, a peptoid, or a small organic molecule. For example, the agent can be an anti-HYAL2 antibody, or a HYAL2 binding fragment of said antibody. It will be recognized that the screening assays of the invention are readily adaptable to a high throughput format. As such, the methods allow for the screening of large numbers of test agents in parallel, and further allow for control reactions to be run in parallel, for example, reactions containing non-inflammatory or anti-inflammatory hyaluronidases, thus providing internal controls useful for confirming that an agent specifically inhibits HYAL2 activity without substantially affecting the activity of hyaluronidases that are not pro-inflammatory.
The present invention further relates to a HYAL2 specific inhibitor identified using such a screening assay. The HYAL2 specific inhibitor can be useful as a purified reagent, for example, as a material to be added to cells in culture to specifically inhibit HYAL2 activity, thus providing a standard for a screening assay to identify agents that can specifically inhibit HYAL2 activity, or can be formulated as a composition, which, for example, can be in a form suitable for administration to a subject, including a vertebrate subject such as a mammal, particularly a human. Such a composition containing a HYAL2 specific inhibitor can be useful for treating an inflammatory disorder associated with HYAL2 activity, for example, a vasculitis, by reducing or inhibiting HYAL2 activity, thereby reducing the generation of the pro-inflammatory 20 kDa intermediate HA breakdown product. Accordingly, the present invention provides a medicament useful for treating a subject having such an inflammatory disorder.
The present invention also relates to a method of ameliorating an inflammatory condition associated with HYAL2 mediated generation of a 20 kDa intermediate HA breakdown product in a subject. Such a method can be performed, for example, by administering a HYAL2 specific inhibitor to the subject, whereby HYAL2 activity is reduced or inhibited, thereby ameliorating the inflammatory condition in the subject. In one embodiment, the HYAL2 specific inhibitor is a HYAL2 specific inhibitor identified according to a screening assay of the invention, for example, an anti-HYAL2 antibody or a HYAL2 binding fragment of said antibody, which specifically binds to and inhibits the activity of HYAL2 without affecting non-inflammatory or anti-inflammatory hyaluronidases, or can be a polynucleotide, peptide, small organic molecule, or the like having HYAL2 specific inhibitory activity. In another embodiment, the HYAL2 specific inhibitor is a polynucleotide that modulates HYAL2 gene expression, for example, an antisense molecule, a ribozyme, a triplexing agent, or an RNA molecule that mediates RNA interference.
The present invention also relates to methods for treating a vasculitis associated with HYAL2 activity in a subject, or a predisposition to such a vasculitis, and further relates to methods of ameliorating such a vasculitis, including preventing or reducing the severity of the vasculitis in a subject. Accordingly, in one embodiment, the present invention relates to a method of ameliorating a vasculitis in a subject, wherein the vasculitis is associated with an elevated (greater than normal) level in the blood of a hyaluronan (HA) catabolite having a molecular mass of about 20 kiloDaltons (kDa) or greater. Such a method can be performed, for example, by inhibiting HYAL2 activity in the subject, without substantially affecting the activity of anti-inflammatory hyaluronidases such as HYAL1, which can degrade pro-inflammatory HA catabolites.
In one aspect of a method of ameliorating a vasculitis in a subject, HYAL2 activity is inhibited in a subject having or predisposed to the vasculitis by administering a HYAL2 specific inhibitor. The inhibitor can be a monoclonal antibody or a small molecule inhibitor. The inhibitor also can be any molecule capable of specifically inhibiting HYAL2, for example, an antibody or other peptide or polypeptide; a small organic molecule such as a peptidomimetic; or a polynucleotide. For example, a specific inhibitor of HYAL2 activity can be a monoclonal antibody that selectively binds HYAL2, but not other hyaluronidases, particularly not anti-inflammatory hyaluronidases. A HYAL2 binding fragment of such a monoclonal antibody, for example, an Fab or F(abxe2x80x2)2 fragment, can be particularly useful for specifically inhibiting HYAL2 activity in vivo because such a fragment does not, for example, stimulate immunoeffector functions such as complement fixation.
In one embodiment, a polynucleotide useful as a specific inhibitor of HYAL2 can be a nucleic acid molecule that is complementary to a naturally occurring polynucleotide encoding HYAL2, including DNA or RNA, thereby reducing or inhibiting the production of HYAL2 polypeptides in a subject. Such nucleic acid molecules can be, for example, antisense nucleic acid molecules, ribozymes, triplexing agents, or RNA molecules that mediate RNA interference. In another embodiment, the nucleic acid molecule for inhibiting HYAL2 activity is a functional nucleic acid that specifically binds to and inhibits HYAL2 activity.
A method of the invention provides a means to prevent or reduce the severity of a vasculitis associated with an elevated level in the blood of a hyaluronan catabolite having a molecular mass of about 20 kDa in a subject by inhibiting HYAL2 activity, thereby reducing or inhibiting generation of the pro-inflammatory 20 kDa HA intermediate degradation product. The vasculitis can be a small vessel vasculitis, which includes vasculitis of small arterioles, capillaries and post-capillary venules; or medium vessel vasculitis, which includes vasculitis of muscular arteries of about 0.2 to 2 mm diameter; or can be a large vessel vasculitis, which includes vasculitis of elastic arteries. For example, the vasculitis can be a vasculitis of the polyarteritis nodosa group of systemic necrotizing vasculitis such as polyarteritis nodosa, which affects medium sized and smaller muscular arteries and, occasionally, larger arteries; or allergic angiitis and granulomatosis (Churg-Strauss variant), which affects small and medium sized arteries, arterioles and veins. The vasculitis also can be a hypersensitivity angiitis, for example, serum sickness, Henoch-Schonlein purpura, vasculitis associated with a connective tissue disorder, or vasculitis associated with essential mixed cryogloulinemia. In addition, the vasculitis can be Wegener granulomatosis, which is a systemic necrotizing vasculitis that generally involves small arteries and veins, particularly in the respiratory tract, kidney and spleen. The vasculitis also can be a giant cell arteritis, for example, temporal arteritis or Takayasu arteritis, or can be Kawasaki disease, thromboangiitis obliterans, or Behcet disease. A subject suitable for treatment according to a method of the invention is one having a vasculitis or a predisposition to a vasculitis due to generation of intermediate HA catabolites by HYAL2 can be identified, for example, by detecting a lower than elevated HYAL2 activity in a blood plasma or serum sample or by detecting the pro-inflammatory HA intermediate, which has a molecular mass of about 20 kDa.